Endothelial cell specification at the osteogenic and angiogenic interface in cranial bone tissue engineering

摘要 由于肿瘤切除、创伤和感染引起的骨丢失的修复和重建仍然是一个重要的问题。 临床挑战全球约有300万例骨科手术使用自体移植或同种异体移植 每年进行一次手术，其中6%是颅颌面手术。骨组织工程已经被誉为 作为替代自体骨修复骨缺损的最终解决方案。然而，长期的成功 骨组织工程的进展受到工程化结构血管化不足的阻碍。当前 目前对组织工程支架血管化的研究进展缓慢，主要是由于我们对组织工程支架血管化的认识不全面 血管生成和血管床在骨修复和再生中的作用。一个功能性的血管网络由 动脉，静脉和毛细血管接口，连接动脉和静脉微血管， 灌注。虽然在早期的动脉和静脉内皮细胞的特化已经得到了很好的研究， 胚胎发育、出生后动脉和静脉扩张的调节以及毛细血管的特化 修复和再生过程中的水平知之甚少。最近的一系列研究表明， 缺氧影响成骨和血管生成界面的内皮细胞（EC）特化， 发育和衰老。缺氧诱导因子-1（HIF-1）通路的基因操作显著提高了 影响毛细血管的特定亚群的形成，称为H型（CD 31 highEmcnhigh）血管， 在长骨干骺端与OSX+成骨细胞偶联。为了更好地理解 缺氧在骨和血管生成界面的修复和再生，我们建立了一系列新的 成像方法允许对毛细血管进行高分辨率、定量和功能分析， 在颅骨缺损部位与Col（I）2.3 GFP+成骨细胞偶联。利用这些新的成像方法， 一层一层使能，纳米纤维介导的颅骨缺损修复模型，我们证明了成骨- 依赖性血管生成由形态和功能上不同的CD 31 +Emcn+和CD 31 +Emcn-组成 船舶.血管类型分布和骨再生检查显示分化 血管生成反应和CD 31 +Emcn+和CD 31 +Emcn-血管的对比分布 Col I（2.3）GFP+成骨细胞、新骨和非骨形成组织，表明EC规格在 毛细血管水平是骨修复和再生中骨生成依赖性血管生成的关键组分。 基于这些发现，我们建议研究缺氧对EC规格的影响以及 在颅骨缺损修复和再生过程中，EC对骨形成的调控异常。三 互补的目标将结合联合收割机成像，遗传和工程方法来定义骨生成- 依赖性EC特化以及缺氧在修复和再生中的作用。我们研究的成功将 为修复中的骨生成和血管生成机制提供了新的见解， 骨再生的翻译靶点。

Abstract Repair and reconstruction of bone loss due to tumor resection, trauma and infection remains a significant clinical challenge. Worldwide, autografts or allografts are used in approximately 3 million orthopaedic procedures annually, of which 6% are craniomaxillofacial in nature. Bone tissue engineering has been hailed as the ultimate solution for replacing bone autograft in repair of bone defects. However, the long-term success of bone tissue engineering is impeded by inadequate vascularization of the engineered construct. The current lack of progress in vascularization of tissue engineered scaffold is attributed to our incomplete understanding of angiogenesis and vascular beds in bone repair and regeneration. A functional blood vessel network consists of arteries, veins and a capillary interface that connects arterial and venous microvessels for proper vascular perfusion. While the specification of arterial and venous endothelium has been well studied during early embryonic development, the postnatal regulation of arterial and venous expansion and specification at capillary level during repair and regeneration is poorly understood. A series of recent studies have suggested that hypoxia affects the endothelial cell (EC) specification at the osteogenic and angiogenic interface in development and aging. Genetic manipulation of the hypoxia inducible factor 1 (HIF-1) pathway markedly affects the formation of specific subsets of capillary vessels, termed Type H (CD31highEmcnhigh) vessels that couple to OSX+ osteoblasts at the long bone metaphysis. To gain a better understanding of the critical role of hypoxia at the osteogenic and angiogenic interface in repair and regeneration, we established a series of novel imaging approaches that permit high resolution, quantitative, and functional analyses of capillary vessels that couple to Col (I) 2.3 GFP+ osteoblasts at a cranial bone defect site. Utilizing these novel imaging approaches in a layer-by-layer enabled, nanofiber-mediated cranial defect repair model, we demonstrate that osteogenesis- dependent angiogenesis consists of morphologically and functionally distinct CD31+Emcn+ and CD31+Emcn- vessels. Examination of blood vessel type distribution and bone regeneration demonstrates differential angiogenic responses and contrasting distributions of CD31+Emcn+ and CD31+Emcn- vessels associated with Col I (2.3) GFP+ osteoblasts, new bone and non-bone forming tissue, suggesting that EC specification at the capillary level is a key component of osteogenesis-dependent angiogenesis in bone repair and regeneration. Based on these findings, we propose to examine the effects of hypoxia on EC specification and the impact of dysregulation of EC specification on bone formation during cranial defect repair and regeneration. Three complementary Aims will combine imaging, genetic and engineering approaches to defining the osteogenesis- dependent EC specification and the role of hypoxia in repair and regeneration. The success of our study will provide novel insights into mechanisms of osteogenesis and angiogenesis in repair, potentially offering novel translational targets for bone regeneration.